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ABNORMAL PRODUCTION OF INTERLEUKIN (IL)-11 AND IL-8 IN POLYCYTHAEMIA VERA
doi:10.1006/cyto.2002.1994
ABNORMAL PRODUCTION OF INTERLEUKIN (IL)-11 AND IL-8 IN POLYCYTHAEMIA VERA Sylvie Hermouet,*1,2 Anne Godard,2 Danielle Pineau,1 Isabelle Corre,2 Sylvie Raher,2 Eric Lippert,2 Yannick Jacques2 We studied the production of interleukin (IL)-11 and IL-8, two cytokines known to affect erythropoiesis, in polycythemia vera (PV). In vivo, IL-11 was detected more frequently in serum and bone marrow (BM) plasma of PV patients than in controls (healthy donors and patients with idiopathic erythrocytosis (IE)). In addition, serum IL-11 levels of PV patients were higher than those of controls. IL-8 was elevated in serum of both PV and IE patients (respective median levels: 38.6 and 242 pg/ml, vs 4.4 pg/ml for healthy donors). BM plasma IL-8 levels of PV patients (508 pg/ml) were significantly higher than those of IE patients (120 pg/ml). In vitro, bone marrow (BM) stromal cells (BMSC) of PV patients produced significantly more IL-11 (6.4) and IL-8 (8.3) than BMSC of healthy donors or IE patients. In conclusion, both IL-11 and IL-8 are overproduced in PV, apparently by BMSC; IL-8 is also overproduced in IE, by cells other than BMSC. 2002 Published by Elsevier Science Ltd. All rights reserved.
Polycythemia vera (PV) is a chronic haematological disorder usually occurring late in life and characterized by an excessive production of mature, fully differentiated erythroid cells originating from a multipotent stem cell and leading to an increased red blood cell mass. The clinical and biological criteria necessary for the diagnosis of PV are now well defined,1–3 but pathogenesis of PV is still for the most part unknown. There is no specific cytogenetic anomaly of BM progenitor cells associated with the disorder. The clinical manifestations and disease severity of PV vary, suggesting that the disorder may arise from different molecular anomalies in different patients. Several alterations of cytokine receptors and signalling molecules have been reported4–6 but none can explain the two main characteristics of PV erythroid progenitors: the ability to generate erythroid colonies in vitro in the absence of exogenous cytokines, notably erythropoietin (EPo) (=endogenous erythroid From the 1Laboratoire d’He´matologie et Groupe ‘‘Cytokines, Re´cepteurs et Transduction de Signal’’, 2INSERM U463, Institut de Biologie des Hoˆpitaux de Nantes, 9 Quai Moncousu, 44093 Nantes Cedex, France Correspondence to: Sylvie Hermouet, Laboratoire d’He´matologie, Institut de Biologie, CHU Nantes, 9 Quai Moncousu, 44093 Nantes Cedex 01, France. Tel: +33 2 40 08 40 51; Fax: +33 2 40 08 41 14; E-mail: [email protected] Received 16 May 2002; revised 13 August 2002; accepted for publication 1 November 2002 1043–4666/02/$-see front matter 2002 Elsevier Science Ltd. All rights reserved. KEY WORDS: polycythaemia vera/interleukin-11/interleukin-8/ EEC/bone marrow stromal cells 178
colony (EEC) formation) and the hypersensitivity to haematopoietic cytokines (SCF, GM-CSF, IL-3, IL-6, EPo) and to IGF-1.7–11 Among the potential mechanisms proposed to explain PV pathogenesis, one involves the autocrine production of a synergizing cytokine rendering erythroid progenitors more responsive to cytokines.5 However, so far there is no evidence of cytokine overproduction in PV. EPo blood levels of PV patients are typically very low12–14 and proliferation of PV erythroid progenitors is usually not blocked by neutralizing antibodies against SCF, GM-CSF, IL-3, IL-6, EPo, singly or in various combinations.5,15 Nonetheless, many studies have shown that in vitro EEC formation depends at least in part on the ‘‘accessory cells’’ present in BM progenitor EEC assays (i.e., stromal and endothelial cells, macrophages, lymphocytes),16 and investigators working with purified PV CD34+ cells often observe a fraction of the EEC obtained in cultures of non-purified BM progenitors. Bone marrow accessory cells are an important source of cytokines involved in erythropoiesis (SCF, GM-CSF, IL-3, IL-6), but not of EPo. BM stromal cells (BMSC) also produce several other molecules capable of altering erythropoiesis.17,18 Two of those, IL-11 and IL-8, have never been studied in PV despite IL-11’s well established stimulating action on erythropoiesis and megakaryocytopoiesis.19 The action of IL-8, which is overproduced in a number of proliferative haematological disorders,20,21 is less well known: high doses may inhibit erythropoiesis, but low doses CYTOKINE, Vol. 20, No. 4 (24 November), 2002: pp 178–183
Abnormal production of interleukin (IL)-11 / 179
TABLE 1.
IL-11 in blood and BM plasma n
n+
(%)
n>30 (pg/ml)
(%)
Median (pg/ml)
Median+ (pg/ml)
Range (pg/ml)
P
Serum Healthy donors IE patients PV patients
38 14 18
7 2 6
(18.4%) (14.3%) (33.3%)
0 0 5*
(27.8%)
0 0 0
2.3 15.5 147.5
0–4 0–25 0–2100
— — <0.05**
BM plasma IE patients PV patients
17 13
2 6
(11.7%) (46.1%)
0 3
(23.1%)
0 0
25 29.5
0–28 0–49
IL-11 was measured in serum and BM plasma using a commercial kit as described in Materials and Methods. IL-11 values are averages of duplicates or triplicates. n: Number of patients tested; n+ (%): number and percentage of patients with detectable IL-11; n>30 (%) : numbers and percentages of patients with >30 pg/ml IL-11 in serum or BM plasma. Median+: median IL-11 value of positive samples only. *P<0.05 compared to healthy donors, Fisher’s exact test. **Compared to healthy donors, Mann–Whitney’s rank sum test.
TABLE 2.
IL-8 in blood and BM plasma n
n+
(%)
n>20 (pg/ml)
(%)
Median (pg/ml)
Range (pg/ml)
P
Serum Healthy donors IE patients PV patients
13 11 18
11 11 18
(84.6%) (100%) (100%)
0 9* 12*
(81.8%) (66.7%)
4.4 242 38.6
0–15.3 2.8–19 820 5–12 248
— <0.001** <0.001**
BM plasma IE patients PV patients
18 10
15 10
(83.3%) (100%)
11 9
(61.1%) (90.0%)
120 508
0–5470 4.8–16 040
— 0.026***
IL-8 was measured in serum and BM plasma using a commercial kit as described in Materials and Methods. IL-8 values are averages of duplicates or triplicates. n: Number of patients tested; n+ (%): number and percentage of patients with detectable IL-8; n>20 (%): numbers and percentages of patients with >20 pg/ml IL-8 in serum or BM plasma. *P<0.05 compared to healthy donors, Fisher’s exact test. **Compared to healthy donors and ***compared to IE patients, Mann–Whitney’s rank sum test.
stimulate the proliferation of CD34+ cells.22–24 Both IL-11 and IL-8 have been shown to synergize with other cytokines.19,23 To investigate whether IL-11 and IL-8 play a role in PV, we studied the levels of IL-11 and IL-8 in serum and in BM plasma of patients diagnosed with PV and in controls: patients with idiopathic erythrocytosis (IE) and healthy donors. Production of IL-11 and IL-8 in cultures of BMSC of PV patients and controls were also studied.
Accepted normal blood serum values for IL-8 are <20 pg/ml; in our series, serum IL-8 levels of all healthy donors were all <16 pg/ml, with a median value of 4.4 pg/ml (Table 2). In contrast, 81.8% of IE patients and 66.7% of PV patients had serum IL-8 levels >20 pg/ml, with medians of 242 pg/ml for IE patients and 38.6 pg/ml for PV patients. In BM plasma, IL-8 levels were significantly higher (by 4.2-fold) for PV patients than for IE patients (BM plasma from healthy donors was not available).
RESULTS
In vitro BMSC production of IL-11 and IL-8
Levels of IL-11 and IL-8 in blood and bone marrow of PV and IE patients In vivo, as previously reported,25 IL-11 was rarely detected in samples of healthy donors and IE patients: for these two groups, less than 20% of blood serum or BM plasma samples were positive for IL-11, and all contained less than 30 pg/ml of IL-11 (Table 1). In contrast, five of 18 PV sera (27.8%) contained more than 30 pg/ml of IL-11. Similarly, 23.1% of PV patients had more than 30 pg/ml of IL-11 in BM plasma, vs none of IE patients and healthy donors.
Because preliminary experiments had determined that basal IL-11 production was below detection level at 24 h, BMSC cytokine production was measured at 48 h. The basal IL-11 production (Table 3) of BMSC of healthy donors and IE patients were always <5 pg/ 1000 cells/48 h. In contrast, basal IL-11 production of BMSC from PV patients was always >5 pg/1000 cells/ 48 h; the median basal production of IL-11 was 6.3fold higher in PV than in IE BMSC cultures. When BMSC cultures were exposed to IL-1, a cytokine commonly used to stimulate cytokine secretion,17,18 production of IL-11 remained significantly higher for
180 / Hermouet et al.
TABLE 3.
CYTOKINE, Vol. 20, No. 4 (24 November, 2002: 178–183)
Basal and IL-1-stimulated production of IL-11 by BM stromal cells IL-11 (pg/1000 cells/48 h Healthy donors
IE patients
PV patients
No stimulation none HD1 HD2 HD3 HD4
1.6 1.2 2.4 1.7
Median Range
1.6 1.2–2.4
IE1 IE5 IE6 IE7 IE9 IE10 Median Range
0 4.4 2.6 1.9 3.6 0.44 2.25 0–4.4
PV2 PV4 PV6 PV7 PV8
6.7 58.7 11.6 16.3 14.3
Median Range
14.3 6.7–58.5 P<0.05*
IL-1 HD1 HD2 HD3 HD4
28.3 21.3 d77 21.4
Median Range
24.8 21.3–d77
IE1 IE5 IE6 IE7 IE9 IE10 Median Range
52.8 13.1 21.6 nd 34.9 nd 28.3 13.1–52.8
PV2 PV4 PV6 PV7 PV8
30.6 91.4 92.8 92.1 7500
Median Range
92.1 30.6–7500 P<0.05**
105 BMMC per well were plated in duplicate or triplicate in 12-well plates, allowed to adhere overnight, then washed free of cells in suspension. After 12 days, adherent cells were washed, 1 ml of fresh medium was added with or without IL-1 (10 ng/ml); 48 h later, supernatants were harvested, centrifuged and stored at 80C; cells were scraped and counted. IL-11 values are expressed as averages of duplicates or triplicates. *P<0.05 compared to healthy donors and IE patients and **P<0.05 compared to IE patients, Mann–Whitney’s rank sum test. nd=No data.
TABLE 4.
Basal and IL-1-stimulated production of IL-8 by BM stromal cells IL-8 (pg/1000 cells/48 h) Healthy donors
IE patients
PV patients
No stimulation none HD1 HD2 HD3 HD4
Median Range
4.2 6.3 10.1 8.3
7.3 4.2–10.1
IE1 IE5 IE6 IE7 IE9 IE10 Median Range
nd 5.8 16.6 4.4 3.7 2.3 4.4 2.3–16.6
PV2 PV4 PV6 PV7 PV8
4.6 83.2 36.3 25.0 99.1
Median Range
36.3 4.6–99.1 P<0.05**
IL-1 HD1 HD2 HD3 HD4
Median Range
624 1397 936 1450
1166 624–1450
IE1 IE5 IE6 IE7 IE9 IE10 Median Range
750 705 2000 nd 1014 nd 882 705–2000
PV2 PV4 PV6 PV7 PV8
1600 294 1935 1721 2818
Median Range
1721 294–2818 P>0.05
105 BMMC per well were plated in duplicate or triplicate in 12-well plates, allowed to adhere overnight, then washed free of cells in suspension. After 12 days, adherent cells were washed, 1 ml of fresh medium was added with or without IL-1 (10 ng/ml); 48 h later, supernatants were harvested, centrifuged and stored at 80C; cells were scraped and counted. IL-8 values are expressed as averages of duplicates or triplicates. *P<0.05 compared to healthy donors and IE patients and **P<0.05 compared to IE patients, Mann–Whitney’s rank sum test. nd=No data.
PV BMSC cultures than for IE BMSC; the response to IL-1 of BMSC of healthy donors and IE patients was comparable (Table 3).
The basal IL-8 production of BMSC from healthy donors and IE patients was always <20 pg/1000 cells/ 48 h (Table 4). In contrast, basal production of BMSC
Abnormal production of interleukin (IL)-11 / 181
TABLE 5.
IL-11 and IL-8 in IE and in PV: summary Serum
Healthy donors IE patients PV patients
BM Plasma
BMSC
IL-11
IL-8
IL-11
IL-8
IL-11
IL-8
Nl Nl >
Nl >> >
nd * *
nd > >>
Nl Nl >
Nl Nl >>
IL-11 values <30 pg/ml and IL-8 values <20 pg/ml in BM plasma and serum were considered normal (Nl) (Tables 1 & 2). BMSC secretion of IL-11 and IL-8 was considered normal when IL-11 was <5 pg/1000 cells/48 h (Table 3) and IL-8 was <20 pg/1000 cells/48 hrs (Table 4). *Similar level in IE and PV patients. nd=No data.
from PV patients was >20 pg/1000 cells/48 h for four patients out of five. Hence, the median basal production of IL-8 was 8.3-fold higher for PV patients than for IE patients. When exposed to IL-1, production of IL-8 remained higher for PV BMSC cultures than for IE BMSC, but the difference was not significant (Table 4). Interestingly, IL-1 stimulated IL-8 production much more efficiently than IL-11 production. PV BMSC responded relatively poorly to IL-1: the median IL-1-induced increases for healthy donors, IE and PV patients were, respectively, 160, 200 and 47-fold for IL-8, and 15.5, 12.6 and 6.4-fold for IL-11.
DISCUSSION The study provides evidence of BM microenvironment dysfunction in PV, a disorder attributed to date solely to abnormalities of erythroid progenitors. Furthermore, PV is the first haematological disease associated with abnormal production of IL-11: a high basal production of IL-11 by BMSC was observed for all PV patients tested. However, IL-11 was significantly elevated in only 30% of PV serum and BM plasma samples. One reason may be that the BMSC culture assay is more sensitive than the serum and BM plasma assays. Indeed, unlike IL-8 which was very stable at 4C or at 80C in both serum and culture supernatants, we found that after thawing, IL-11 was unstable in serum or BM plasma and tended to become rapidly degraded (unpublished observation); in contrast, IL-11 was stable in culture supernatants. Another reason could be that most of the IL-11 produced by PV BMSC is used locally by BM progenitors and that only a small fraction reaches peripheral blood. However, given the heterogeneity of PV clinical presentation and disease outcome, it is probable that PV has several different causes and thus not all PV patients overproduce IL-11. In our series, approximately 30% of patients diagnosed with PV, with a positive EEC assay, presented no evidence in serum or BM plasma of IL-11/IL-8 overproduction. Excessive production of IL-11 appears to be specific
of PV: preliminary studies of serum and BM plasma of patients with essential thrombocytemia, another myeloproliferative disorder, showed no evidence of abnormal IL-11 production (unpublished observations). In addition, the study confirms the association of proliferative haematological disorders, malignant or benign, with high IL-8 production: patients with erythrocytosis, primary (PV) or idiopathic, have high serum IL-8, an abnormality previously reported for acute lymphoid and myeloid leukaemia, chronic Bcell lymphoid leukaemia and non-Hodgkin lymphoma.20,21,24,26 IL-8, mostly known as a powerful neutrophil attractant, was initially described as an autocrine growth factor for melanoma cells; its stimulating effect on proliferation is well established for diverse cell types.24,27,28 In the haematopoietic system, the effect of IL-8 on cell proliferation is less well defined: used in vitro at very high doses (>50 ng/ml), IL-8 inhibits erythroid colony formation.22 However, the highest concentrations of IL-8 observed in serum and BM plasma of patients were all <20 ng/ml; standing by others and ourselves have shown that when its concentration range is within 1 to 10 or 20 ng/ml, IL-8 stimulates CD34+ cell proliferation and increases cell survival.23,24,28 One could argue that in the haematopoietic system as in other cell systems, different causes in different pathologies may induce a common cellular response (an increased production of IL-8) with the apparent consequence of facilitating cell proliferation. Because of the large range of cells expressing IL-8 receptors, an increased production of IL-8 would be expected to enhance survival or/and proliferation of haematopoietic cells of different lineages. The cellular source of abnormal IL-8 production seems to vary: the malignant cells themselves in acute and chronic lymphocytic leukaemia,20,26 cells of the BM microenvironment (BMSC) in PV. As summarized in Table 5, PV patients exhibit high BMSC production and, logically, high BM plasma levels and moderate IL-8 levels in blood serum. Inversely, for IE patients the highest IL-8 levels are found in serum, IL-8 BM plasma are moderately high
182 / Hermouet et al.
and IL-8 production of IE BMSC is normal, suggesting that the cells responsible for the elevation of serum IL-8 are not in the bone marrow, but peripheral, and that the IL-8 found in the BM plasma of IE patients could be contamination from blood. Because both IL-8 and IL-11 act in synergy with other haematopoietic cytokines,19,23 overproduction of IL-8 and IL-11 by PV BMSC could explain one characteristic of PV progenitors, hypersensitivity to cytokines. But it appears that EEC formation, the other characteristic of PV, is not explained by high levels of IL-11 and IL-8: normal and IE BMMC grown in vitro in the presence of various concentrations of IL-11 and IL-8, alone or together, did not form EEC (unpublished observations). In this respect, further studies investigating IL-11 and IL-8 receptor expression by PV progenitors, as well as whether PV progenitors (CD34+ cells or subsets of CD34+ cells) secrete IL-11 in an autocrine fashion, should be very informative. Normal CD34+ cells are known to secrete numerous cytokines, and abnormal cytokine secretion by CD34+ cells is already demonstrated for one myeloproliferative syndrome, chronic myeloid leukaemia.29,30 Other unanswered questions concern the causes and mechanisms of dysregulation of IL-8 production in PV, which may or may not be common to other haematological disorders.24 The cause(s) and mechanisms of the specific dysregulation of IL-11 expression in PV also need to be investigated. Last, we cannot exclude that the causes responsible for the excessive production of IL-8 and IL-11 by PV BM stromal cells have other consequences, for example the abnormal secretion by BM progenitors or stromal cells of additional molecules acting as stimulants of erythropoiesis. In conclusion, IL-8 is produced in excess by the majority of IE and PV patients; PV patients also overproduce IL-11. As constant BM progenitor cell stimulation by IL-11 and IL-8 could explain the hypersensitivity of PV progenitors to haematopoietic cytokines, further investigation of the cellular sources and mechanisms of dysregulation of IL-11 and IL-8 production should provide novel insights into PV pathogenesis.
CYTOKINE, Vol. 20, No. 4 (24 November, 2002: 178–183)
secondary erythrocytosis, no diagnostic features of PV; all IE patients in the study had a negative EEC assay).2 Serum was also obtained from healthy donors; samples of normal BM were obtained from allograft donors. Due to incomplete collection or insufficient sample quantity, all of the assays described in the study could not always be performed for each patient; BM plasma from healthy donors was not available.
IL-11 and IL-8 assays Blood samples were collected in dry vacutainer tubes, centrifuged at 300g for 15 min, then the supernatant (serum) was pipetted and aliquoted. Bone marrow samples were collected in tubes containing heparin, centrifuged at 150g for 10 min without brake, then the supernatant (BM plasma) was pipetted carefully so as not to disturb the cell pellet, and aliquoted. Aliquots of blood serum and BM plasma were frozen at 80C until use.13 IL-11 and IL-8 were measured in duplicate or triplicate using the Quantikine IVD IL-11 kit (R & D, Abingdon, UK) and IL-8 EASIA kit (Biosource Europe). Values (averages of duplicates or triplicates) are expressed as pg/ml (blood serum, BM plasma) or pg/1000 cells (48 h BMSC culture supernatants). Minimum detectable concentrations were 1 pg/ml for IL-11 and 0.7 pg/ ml for IL-8.
BMSC culture 105 BMMC in 1 ml medium (RPMI+10% fetal bovine serum or FBS) per well were plated in duplicate or triplicate in 12-well plates. Cells were allowed to adhere overnight, then were washed free of suspension cells and grown for 12 days. On day 12, adherent cells were washed and 1 ml of fresh medium was added to each well, with or without IL-1(10 ng/ml). 48 h later, supernatants were harvested, centrifuged and stored at 80C; cells were scraped and counted.
Statistical analysis For analysis of serum and BM plasma data of groups of patients or healthy donors, Mann–Whitney’s rank sum test was used (mean values differed from median values); Fisher’s exact test was used to compare ratios. A P value <0.05 was considered statistically significant.
Acknowledgements MATERIALS AND METHODS Patients Before any treatment and with informed consent, blood and BM samples were obtained from patients with erythrocytosis (packed cell volume >0.51 in men, >0.48 in women) and diagnosed according to standard diagnostic critieria with PV (all PV patients in this study had a positive EEC assay) or IE (absolute increase in red cell mass, no known cause of
We thank Mrs Annie Alle´graud, Mrs Ire`ne Dobo, Dr Nathalie Boiret, Dr Franc¸ois Girodon and Dr Pascal Mossuz (respectively, Centre Hospitaliers Universitaires de Limoges, Angers, Clermont-Ferrand, Dijon and Grenoble, France) for providing samples of serum and BM plasma. The study was supported by grants from the Ligue Nationale contre le Cancer (Comite´ de Loire-Atlantique) and the Association pour la Recherche contre le Cancer (ARC).
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ABNORMAL PRODUCTION OF INTERLEUKIN (IL)-11 AND IL-8 IN POLYCYTHAEMIA VERA Sylvie Hermouet,*1,2 Anne Godard,2 Danielle Pineau,1 Isabelle Corre,2 Sylvie Raher,2 Eric Lippert,2 Yannick Jacques2 We studied the production of interleukin (IL)-11 and IL-8, two cytokines known to affect erythropoiesis, in polycythemia vera (PV). In vivo, IL-11 was detected more frequently in serum and bone marrow (BM) plasma of PV patients than in controls (healthy donors and patients with idiopathic erythrocytosis (IE)). In addition, serum IL-11 levels of PV patients were higher than those of controls. IL-8 was elevated in serum of both PV and IE patients (respective median levels: 38.6 and 242 pg/ml, vs 4.4 pg/ml for healthy donors). BM plasma IL-8 levels of PV patients (508 pg/ml) were significantly higher than those of IE patients (120 pg/ml). In vitro, bone marrow (BM) stromal cells (BMSC) of PV patients produced significantly more IL-11 (6.4) and IL-8 (8.3) than BMSC of healthy donors or IE patients. In conclusion, both IL-11 and IL-8 are overproduced in PV, apparently by BMSC; IL-8 is also overproduced in IE, by cells other than BMSC. 2002 Published by Elsevier Science Ltd. All rights reserved.
Polycythemia vera (PV) is a chronic haematological disorder usually occurring late in life and characterized by an excessive production of mature, fully differentiated erythroid cells originating from a multipotent stem cell and leading to an increased red blood cell mass. The clinical and biological criteria necessary for the diagnosis of PV are now well defined,1–3 but pathogenesis of PV is still for the most part unknown. There is no specific cytogenetic anomaly of BM progenitor cells associated with the disorder. The clinical manifestations and disease severity of PV vary, suggesting that the disorder may arise from different molecular anomalies in different patients. Several alterations of cytokine receptors and signalling molecules have been reported4–6 but none can explain the two main characteristics of PV erythroid progenitors: the ability to generate erythroid colonies in vitro in the absence of exogenous cytokines, notably erythropoietin (EPo) (=endogenous erythroid From the 1Laboratoire d’He´matologie et Groupe ‘‘Cytokines, Re´cepteurs et Transduction de Signal’’, 2INSERM U463, Institut de Biologie des Hoˆpitaux de Nantes, 9 Quai Moncousu, 44093 Nantes Cedex, France Correspondence to: Sylvie Hermouet, Laboratoire d’He´matologie, Institut de Biologie, CHU Nantes, 9 Quai Moncousu, 44093 Nantes Cedex 01, France. Tel: +33 2 40 08 40 51; Fax: +33 2 40 08 41 14; E-mail: [email protected] Received 16 May 2002; revised 13 August 2002; accepted for publication 1 November 2002 1043–4666/02/$-see front matter 2002 Elsevier Science Ltd. All rights reserved. KEY WORDS: polycythaemia vera/interleukin-11/interleukin-8/ EEC/bone marrow stromal cells 178
colony (EEC) formation) and the hypersensitivity to haematopoietic cytokines (SCF, GM-CSF, IL-3, IL-6, EPo) and to IGF-1.7–11 Among the potential mechanisms proposed to explain PV pathogenesis, one involves the autocrine production of a synergizing cytokine rendering erythroid progenitors more responsive to cytokines.5 However, so far there is no evidence of cytokine overproduction in PV. EPo blood levels of PV patients are typically very low12–14 and proliferation of PV erythroid progenitors is usually not blocked by neutralizing antibodies against SCF, GM-CSF, IL-3, IL-6, EPo, singly or in various combinations.5,15 Nonetheless, many studies have shown that in vitro EEC formation depends at least in part on the ‘‘accessory cells’’ present in BM progenitor EEC assays (i.e., stromal and endothelial cells, macrophages, lymphocytes),16 and investigators working with purified PV CD34+ cells often observe a fraction of the EEC obtained in cultures of non-purified BM progenitors. Bone marrow accessory cells are an important source of cytokines involved in erythropoiesis (SCF, GM-CSF, IL-3, IL-6), but not of EPo. BM stromal cells (BMSC) also produce several other molecules capable of altering erythropoiesis.17,18 Two of those, IL-11 and IL-8, have never been studied in PV despite IL-11’s well established stimulating action on erythropoiesis and megakaryocytopoiesis.19 The action of IL-8, which is overproduced in a number of proliferative haematological disorders,20,21 is less well known: high doses may inhibit erythropoiesis, but low doses CYTOKINE, Vol. 20, No. 4 (24 November), 2002: pp 178–183
Abnormal production of interleukin (IL)-11 / 179
TABLE 1.
IL-11 in blood and BM plasma n
n+
(%)
n>30 (pg/ml)
(%)
Median (pg/ml)
Median+ (pg/ml)
Range (pg/ml)
P
Serum Healthy donors IE patients PV patients
38 14 18
7 2 6
(18.4%) (14.3%) (33.3%)
0 0 5*
(27.8%)
0 0 0
2.3 15.5 147.5
0–4 0–25 0–2100
— — <0.05**
BM plasma IE patients PV patients
17 13
2 6
(11.7%) (46.1%)
0 3
(23.1%)
0 0
25 29.5
0–28 0–49
IL-11 was measured in serum and BM plasma using a commercial kit as described in Materials and Methods. IL-11 values are averages of duplicates or triplicates. n: Number of patients tested; n+ (%): number and percentage of patients with detectable IL-11; n>30 (%) : numbers and percentages of patients with >30 pg/ml IL-11 in serum or BM plasma. Median+: median IL-11 value of positive samples only. *P<0.05 compared to healthy donors, Fisher’s exact test. **Compared to healthy donors, Mann–Whitney’s rank sum test.
TABLE 2.
IL-8 in blood and BM plasma n
n+
(%)
n>20 (pg/ml)
(%)
Median (pg/ml)
Range (pg/ml)
P
Serum Healthy donors IE patients PV patients
13 11 18
11 11 18
(84.6%) (100%) (100%)
0 9* 12*
(81.8%) (66.7%)
4.4 242 38.6
0–15.3 2.8–19 820 5–12 248
— <0.001** <0.001**
BM plasma IE patients PV patients
18 10
15 10
(83.3%) (100%)
11 9
(61.1%) (90.0%)
120 508
0–5470 4.8–16 040
— 0.026***
IL-8 was measured in serum and BM plasma using a commercial kit as described in Materials and Methods. IL-8 values are averages of duplicates or triplicates. n: Number of patients tested; n+ (%): number and percentage of patients with detectable IL-8; n>20 (%): numbers and percentages of patients with >20 pg/ml IL-8 in serum or BM plasma. *P<0.05 compared to healthy donors, Fisher’s exact test. **Compared to healthy donors and ***compared to IE patients, Mann–Whitney’s rank sum test.
stimulate the proliferation of CD34+ cells.22–24 Both IL-11 and IL-8 have been shown to synergize with other cytokines.19,23 To investigate whether IL-11 and IL-8 play a role in PV, we studied the levels of IL-11 and IL-8 in serum and in BM plasma of patients diagnosed with PV and in controls: patients with idiopathic erythrocytosis (IE) and healthy donors. Production of IL-11 and IL-8 in cultures of BMSC of PV patients and controls were also studied.
Accepted normal blood serum values for IL-8 are <20 pg/ml; in our series, serum IL-8 levels of all healthy donors were all <16 pg/ml, with a median value of 4.4 pg/ml (Table 2). In contrast, 81.8% of IE patients and 66.7% of PV patients had serum IL-8 levels >20 pg/ml, with medians of 242 pg/ml for IE patients and 38.6 pg/ml for PV patients. In BM plasma, IL-8 levels were significantly higher (by 4.2-fold) for PV patients than for IE patients (BM plasma from healthy donors was not available).
RESULTS
In vitro BMSC production of IL-11 and IL-8
Levels of IL-11 and IL-8 in blood and bone marrow of PV and IE patients In vivo, as previously reported,25 IL-11 was rarely detected in samples of healthy donors and IE patients: for these two groups, less than 20% of blood serum or BM plasma samples were positive for IL-11, and all contained less than 30 pg/ml of IL-11 (Table 1). In contrast, five of 18 PV sera (27.8%) contained more than 30 pg/ml of IL-11. Similarly, 23.1% of PV patients had more than 30 pg/ml of IL-11 in BM plasma, vs none of IE patients and healthy donors.
Because preliminary experiments had determined that basal IL-11 production was below detection level at 24 h, BMSC cytokine production was measured at 48 h. The basal IL-11 production (Table 3) of BMSC of healthy donors and IE patients were always <5 pg/ 1000 cells/48 h. In contrast, basal IL-11 production of BMSC from PV patients was always >5 pg/1000 cells/ 48 h; the median basal production of IL-11 was 6.3fold higher in PV than in IE BMSC cultures. When BMSC cultures were exposed to IL-1, a cytokine commonly used to stimulate cytokine secretion,17,18 production of IL-11 remained significantly higher for
180 / Hermouet et al.
TABLE 3.
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Basal and IL-1-stimulated production of IL-11 by BM stromal cells IL-11 (pg/1000 cells/48 h Healthy donors
IE patients
PV patients
No stimulation none HD1 HD2 HD3 HD4
1.6 1.2 2.4 1.7
Median Range
1.6 1.2–2.4
IE1 IE5 IE6 IE7 IE9 IE10 Median Range
0 4.4 2.6 1.9 3.6 0.44 2.25 0–4.4
PV2 PV4 PV6 PV7 PV8
6.7 58.7 11.6 16.3 14.3
Median Range
14.3 6.7–58.5 P<0.05*
IL-1 HD1 HD2 HD3 HD4
28.3 21.3 d77 21.4
Median Range
24.8 21.3–d77
IE1 IE5 IE6 IE7 IE9 IE10 Median Range
52.8 13.1 21.6 nd 34.9 nd 28.3 13.1–52.8
PV2 PV4 PV6 PV7 PV8
30.6 91.4 92.8 92.1 7500
Median Range
92.1 30.6–7500 P<0.05**
105 BMMC per well were plated in duplicate or triplicate in 12-well plates, allowed to adhere overnight, then washed free of cells in suspension. After 12 days, adherent cells were washed, 1 ml of fresh medium was added with or without IL-1 (10 ng/ml); 48 h later, supernatants were harvested, centrifuged and stored at 80C; cells were scraped and counted. IL-11 values are expressed as averages of duplicates or triplicates. *P<0.05 compared to healthy donors and IE patients and **P<0.05 compared to IE patients, Mann–Whitney’s rank sum test. nd=No data.
TABLE 4.
Basal and IL-1-stimulated production of IL-8 by BM stromal cells IL-8 (pg/1000 cells/48 h) Healthy donors
IE patients
PV patients
No stimulation none HD1 HD2 HD3 HD4
Median Range
4.2 6.3 10.1 8.3
7.3 4.2–10.1
IE1 IE5 IE6 IE7 IE9 IE10 Median Range
nd 5.8 16.6 4.4 3.7 2.3 4.4 2.3–16.6
PV2 PV4 PV6 PV7 PV8
4.6 83.2 36.3 25.0 99.1
Median Range
36.3 4.6–99.1 P<0.05**
IL-1 HD1 HD2 HD3 HD4
Median Range
624 1397 936 1450
1166 624–1450
IE1 IE5 IE6 IE7 IE9 IE10 Median Range
750 705 2000 nd 1014 nd 882 705–2000
PV2 PV4 PV6 PV7 PV8
1600 294 1935 1721 2818
Median Range
1721 294–2818 P>0.05
105 BMMC per well were plated in duplicate or triplicate in 12-well plates, allowed to adhere overnight, then washed free of cells in suspension. After 12 days, adherent cells were washed, 1 ml of fresh medium was added with or without IL-1 (10 ng/ml); 48 h later, supernatants were harvested, centrifuged and stored at 80C; cells were scraped and counted. IL-8 values are expressed as averages of duplicates or triplicates. *P<0.05 compared to healthy donors and IE patients and **P<0.05 compared to IE patients, Mann–Whitney’s rank sum test. nd=No data.
PV BMSC cultures than for IE BMSC; the response to IL-1 of BMSC of healthy donors and IE patients was comparable (Table 3).
The basal IL-8 production of BMSC from healthy donors and IE patients was always <20 pg/1000 cells/ 48 h (Table 4). In contrast, basal production of BMSC
Abnormal production of interleukin (IL)-11 / 181
TABLE 5.
IL-11 and IL-8 in IE and in PV: summary Serum
Healthy donors IE patients PV patients
BM Plasma
BMSC
IL-11
IL-8
IL-11
IL-8
IL-11
IL-8
Nl Nl >
Nl >> >
nd * *
nd > >>
Nl Nl >
Nl Nl >>
IL-11 values <30 pg/ml and IL-8 values <20 pg/ml in BM plasma and serum were considered normal (Nl) (Tables 1 & 2). BMSC secretion of IL-11 and IL-8 was considered normal when IL-11 was <5 pg/1000 cells/48 h (Table 3) and IL-8 was <20 pg/1000 cells/48 hrs (Table 4). *Similar level in IE and PV patients. nd=No data.
from PV patients was >20 pg/1000 cells/48 h for four patients out of five. Hence, the median basal production of IL-8 was 8.3-fold higher for PV patients than for IE patients. When exposed to IL-1, production of IL-8 remained higher for PV BMSC cultures than for IE BMSC, but the difference was not significant (Table 4). Interestingly, IL-1 stimulated IL-8 production much more efficiently than IL-11 production. PV BMSC responded relatively poorly to IL-1: the median IL-1-induced increases for healthy donors, IE and PV patients were, respectively, 160, 200 and 47-fold for IL-8, and 15.5, 12.6 and 6.4-fold for IL-11.
DISCUSSION The study provides evidence of BM microenvironment dysfunction in PV, a disorder attributed to date solely to abnormalities of erythroid progenitors. Furthermore, PV is the first haematological disease associated with abnormal production of IL-11: a high basal production of IL-11 by BMSC was observed for all PV patients tested. However, IL-11 was significantly elevated in only 30% of PV serum and BM plasma samples. One reason may be that the BMSC culture assay is more sensitive than the serum and BM plasma assays. Indeed, unlike IL-8 which was very stable at 4C or at 80C in both serum and culture supernatants, we found that after thawing, IL-11 was unstable in serum or BM plasma and tended to become rapidly degraded (unpublished observation); in contrast, IL-11 was stable in culture supernatants. Another reason could be that most of the IL-11 produced by PV BMSC is used locally by BM progenitors and that only a small fraction reaches peripheral blood. However, given the heterogeneity of PV clinical presentation and disease outcome, it is probable that PV has several different causes and thus not all PV patients overproduce IL-11. In our series, approximately 30% of patients diagnosed with PV, with a positive EEC assay, presented no evidence in serum or BM plasma of IL-11/IL-8 overproduction. Excessive production of IL-11 appears to be specific
of PV: preliminary studies of serum and BM plasma of patients with essential thrombocytemia, another myeloproliferative disorder, showed no evidence of abnormal IL-11 production (unpublished observations). In addition, the study confirms the association of proliferative haematological disorders, malignant or benign, with high IL-8 production: patients with erythrocytosis, primary (PV) or idiopathic, have high serum IL-8, an abnormality previously reported for acute lymphoid and myeloid leukaemia, chronic Bcell lymphoid leukaemia and non-Hodgkin lymphoma.20,21,24,26 IL-8, mostly known as a powerful neutrophil attractant, was initially described as an autocrine growth factor for melanoma cells; its stimulating effect on proliferation is well established for diverse cell types.24,27,28 In the haematopoietic system, the effect of IL-8 on cell proliferation is less well defined: used in vitro at very high doses (>50 ng/ml), IL-8 inhibits erythroid colony formation.22 However, the highest concentrations of IL-8 observed in serum and BM plasma of patients were all <20 ng/ml; standing by others and ourselves have shown that when its concentration range is within 1 to 10 or 20 ng/ml, IL-8 stimulates CD34+ cell proliferation and increases cell survival.23,24,28 One could argue that in the haematopoietic system as in other cell systems, different causes in different pathologies may induce a common cellular response (an increased production of IL-8) with the apparent consequence of facilitating cell proliferation. Because of the large range of cells expressing IL-8 receptors, an increased production of IL-8 would be expected to enhance survival or/and proliferation of haematopoietic cells of different lineages. The cellular source of abnormal IL-8 production seems to vary: the malignant cells themselves in acute and chronic lymphocytic leukaemia,20,26 cells of the BM microenvironment (BMSC) in PV. As summarized in Table 5, PV patients exhibit high BMSC production and, logically, high BM plasma levels and moderate IL-8 levels in blood serum. Inversely, for IE patients the highest IL-8 levels are found in serum, IL-8 BM plasma are moderately high
182 / Hermouet et al.
and IL-8 production of IE BMSC is normal, suggesting that the cells responsible for the elevation of serum IL-8 are not in the bone marrow, but peripheral, and that the IL-8 found in the BM plasma of IE patients could be contamination from blood. Because both IL-8 and IL-11 act in synergy with other haematopoietic cytokines,19,23 overproduction of IL-8 and IL-11 by PV BMSC could explain one characteristic of PV progenitors, hypersensitivity to cytokines. But it appears that EEC formation, the other characteristic of PV, is not explained by high levels of IL-11 and IL-8: normal and IE BMMC grown in vitro in the presence of various concentrations of IL-11 and IL-8, alone or together, did not form EEC (unpublished observations). In this respect, further studies investigating IL-11 and IL-8 receptor expression by PV progenitors, as well as whether PV progenitors (CD34+ cells or subsets of CD34+ cells) secrete IL-11 in an autocrine fashion, should be very informative. Normal CD34+ cells are known to secrete numerous cytokines, and abnormal cytokine secretion by CD34+ cells is already demonstrated for one myeloproliferative syndrome, chronic myeloid leukaemia.29,30 Other unanswered questions concern the causes and mechanisms of dysregulation of IL-8 production in PV, which may or may not be common to other haematological disorders.24 The cause(s) and mechanisms of the specific dysregulation of IL-11 expression in PV also need to be investigated. Last, we cannot exclude that the causes responsible for the excessive production of IL-8 and IL-11 by PV BM stromal cells have other consequences, for example the abnormal secretion by BM progenitors or stromal cells of additional molecules acting as stimulants of erythropoiesis. In conclusion, IL-8 is produced in excess by the majority of IE and PV patients; PV patients also overproduce IL-11. As constant BM progenitor cell stimulation by IL-11 and IL-8 could explain the hypersensitivity of PV progenitors to haematopoietic cytokines, further investigation of the cellular sources and mechanisms of dysregulation of IL-11 and IL-8 production should provide novel insights into PV pathogenesis.
CYTOKINE, Vol. 20, No. 4 (24 November, 2002: 178–183)
secondary erythrocytosis, no diagnostic features of PV; all IE patients in the study had a negative EEC assay).2 Serum was also obtained from healthy donors; samples of normal BM were obtained from allograft donors. Due to incomplete collection or insufficient sample quantity, all of the assays described in the study could not always be performed for each patient; BM plasma from healthy donors was not available.
IL-11 and IL-8 assays Blood samples were collected in dry vacutainer tubes, centrifuged at 300g for 15 min, then the supernatant (serum) was pipetted and aliquoted. Bone marrow samples were collected in tubes containing heparin, centrifuged at 150g for 10 min without brake, then the supernatant (BM plasma) was pipetted carefully so as not to disturb the cell pellet, and aliquoted. Aliquots of blood serum and BM plasma were frozen at 80C until use.13 IL-11 and IL-8 were measured in duplicate or triplicate using the Quantikine IVD IL-11 kit (R & D, Abingdon, UK) and IL-8 EASIA kit (Biosource Europe). Values (averages of duplicates or triplicates) are expressed as pg/ml (blood serum, BM plasma) or pg/1000 cells (48 h BMSC culture supernatants). Minimum detectable concentrations were 1 pg/ml for IL-11 and 0.7 pg/ ml for IL-8.
BMSC culture 105 BMMC in 1 ml medium (RPMI+10% fetal bovine serum or FBS) per well were plated in duplicate or triplicate in 12-well plates. Cells were allowed to adhere overnight, then were washed free of suspension cells and grown for 12 days. On day 12, adherent cells were washed and 1 ml of fresh medium was added to each well, with or without IL-1(10 ng/ml). 48 h later, supernatants were harvested, centrifuged and stored at 80C; cells were scraped and counted.
Statistical analysis For analysis of serum and BM plasma data of groups of patients or healthy donors, Mann–Whitney’s rank sum test was used (mean values differed from median values); Fisher’s exact test was used to compare ratios. A P value <0.05 was considered statistically significant.
Acknowledgements MATERIALS AND METHODS Patients Before any treatment and with informed consent, blood and BM samples were obtained from patients with erythrocytosis (packed cell volume >0.51 in men, >0.48 in women) and diagnosed according to standard diagnostic critieria with PV (all PV patients in this study had a positive EEC assay) or IE (absolute increase in red cell mass, no known cause of
We thank Mrs Annie Alle´graud, Mrs Ire`ne Dobo, Dr Nathalie Boiret, Dr Franc¸ois Girodon and Dr Pascal Mossuz (respectively, Centre Hospitaliers Universitaires de Limoges, Angers, Clermont-Ferrand, Dijon and Grenoble, France) for providing samples of serum and BM plasma. The study was supported by grants from the Ligue Nationale contre le Cancer (Comite´ de Loire-Atlantique) and the Association pour la Recherche contre le Cancer (ARC).
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